A major focus of cellular and molecular research has concentrated on developing means to regulate gene expression (i.e., gene transcription and translation) in an effort to treat and cure a variety of disease and conditions. It is hoped that the up- or down-regulation of specific genes will alter or circumvent the molecular mechanisms underlying these diseases and conditions. The importance of such research has dramatically increased as the Human Genome Project continues to identify genes at an accelerated pace. Gene identification alone, however, is only a preliminary step towards gaining control over the associated diseases and conditions. Methods to manipulate the expression of these newly identified genes are needed as well.
Currently, several general methods have been developed to regulate and control gene expression at either the transcriptional or translational steps. Each of these methods suffers from significant drawbacks.
A. Global Transcription and Translation Regulators
One means of regulating gene expression is to use chemicals that alter the expression of all genes within a cell, tissue, or organism. For example, cycloheximide blocks the peptidyl transferase reaction on eukaryotic ribosomes and acts as a general inhibitor of translation (i.e., the translation of all genes within treated cells is inhibited). Likewise, .alpha.-amantin globally blocks mRNA synthesis by binding to eukaryotic RNA polymerase II. Furthermore, actinomycin D is capable of blocking RNA synthesis by intercalating into guanine-cytosine base pairs and disrupting transcription; netropsin and distamycin A block transcription by binding to DNA and blocking RNA polymerase; and acridines, such as proflavine, inhibit RNA synthesis by blocking the formation of the DNA/RNA polymerase complex. Because these chemicals prevent the expression of all genes, any prolonged treatment results in the loss of critical factors needed to maintain the cells, leading to irreparable damage or cell death (e.g., .alpha.-amantin was originally identified as a potent poison from the mushroom Amanita phalloides; Wieland and Faulstich, Crit. Rev. B. 5: 185 [1978]). To overcome these drawbacks, methods of regulating the expression of specific genes or gene families must be developed.
B. Regulation of Signal Transduction Pathways
One means of regulating gene expression is to activate or repress the signal transduction pathways that are responsible for regulating gene transcription. By activating or inhibiting important steps in the pathways (e.g., binding of signalling molecules to receptors, entry of signalling molecules into cells or nuclei, covalent modification of enzymes, or release or sequestration of ions from organelles), gene expression can be activated or repressed. For example, pain relievers such as aspirin and ibuprofen inhibit the enzymatic production of prostaglandins and result in decreased swelling and inflammation brought about by the signalling pathways normally initiated by the prostaglandins.
Unfortunately, the regulation of signal transduction pathways is not a viable means of treating many diseases and conditions. Most pathways have not been sufficiently characterized to rationally develop means of regulating expression and treating disease while avoiding unwanted side-effects. For example, many signal transduction pathways regulate a variety of genes in a variety of different cell types. Thus, in an attempt to shut off a gene responsible for a given disease, the pathway may also down-regulate other genes responsible for critical metabolic processes in the cells. Also, many signalling pathways are redundant (i.e., more than one pathway controls the down-stream regulatory event). Thus, by activating or repressing one pathway, another may compensate and confound the attempt at controlling gene expression. Furthermore, many signal transduction pathways cross-talk (i.e., share similar components and co-regulate one another). Thus the regulation of one pathway may result in the undesired regulation of other known, and yet unidentified, pathways. By inhibiting or activating a given step within a pathway, a range of known or unknown side-effects can occur. For example, prostaglandin signalling is involved not only in inflammatory responses, but is also believed to be involved in platelet aggregation, the sleep-wake cycle, some aspects of vision, luteolysis, and any number of yet unidentified physiological effects. Thus, in general, the regulation of signal transduction pathways provides a too broad and unpredictable means for controlling gene expression.
C. Gene Therapy
With the development of gene therapy techniques, it has become possible to replace or insert genes of interest into organisms. In theory, overactively expressed or mutated genes can be replaced by "normal" copies. Also, genes can be linked to controllable promoter elements (i.e., a promoter that can be turned on or off by administration of appropriate signalling compounds) and can be placed into target cells. For example, the gene for a desired transcription factor could be placed under the control of such an inducible/repressible promoter. Using this technique, gene families that are activated or repressed by the transcription factor can be coordinately regulated by the administration of the appropriate signalling compounds. These transcription factors can be wild-type (i.e., to directly activate or repress a gene), mutants with DNA binding capability but altered active sites (i.e., to compete with the cell's natural transcription factors for binding to gene enhancers), or mutants with wild-type heterodimerization domains but altered active sites or DNA binding sites (i.e., to bind to the cell's natural transcription factors and prevent it from binding to enhancers and regulating gene expression).
Unfortunately, gene therapy techniques, as described above, are only in their initial stages of development. There are still significant problems to overcome, such as the lack of efficient delivery systems, lack of sustained expression, and host immune reactions (Verma and Somia, Nature 389, 239 [1997]). Even if these technologies eventually become widely available, they will be extremely complex, time-consuming, and unpredictable.
The art remains in need of means for regulating gene expression to control and treat human diseases such as cancer and viral infections. Such an approach should repress or activate specific genes or gene families without producing harmful side effects.